Abstract

The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown–Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development.

Introduction

Chronic inflammatory disorders are multifactorial diseases whose development depends on a combination of environmental triggers and genetic risk factors (1). To date, most genetic studies performed in human populations have used a single-locus analysis strategy, allowing the association of gene variants with a specific phenotype. This has led to the identification of a great number of gene variants, most of them conferring only a small risk increment. In most cases, the identified gene variants explain <20% of disease heritability (2). One potential source of this low heritability rate may result from interactions between polymorphic genes, also known as epistasis, that are not taken into account by the current methods of analysis (2–4). Indeed, many susceptibility genes may exhibit effects that are totally or partially dependent on interactions with other genes (5). This highlights the importance of integrating gene–gene interactions in the genetic studies. In this regard, mouse and rat models of human diseases represent invaluable tools not only for the identification of gene–gene interactions involved in susceptibility to immune-mediated diseases (6) but also for deciphering the mechanisms whereby these interactions affect the disease pathophysiology.

Signaling through the TCR directs the functional outcome of T cell activation. Thus, because TCR signaling integrity controls T cell functions, any polymorphism or deficiency for TCR signaling genes could predispose to immune-mediated diseases. Indeed, our previous genetic studies using animal models have independently identified the role of the TCR signaling genes Vav1 (7–9) and Themis1 (10) in the susceptibility to autoimmune and inflammatory diseases. Vav1 is a guanine nucleotide exchange factor (GEF) for Rho GTPases, which plays a nonredundant role in transducing TCR signals important for T cell development and functions (11–14). Vav1 phosphorylation relieves its catalytic domain and causes Vav1 to promote GDP-GTP exchange on the Rho, Rac1, and Cdc42 small GTPases. Besides this well-documented GEF activity, Vav1 also plays a role as an adaptor protein. Indeed, the generation of mice that express a mutant Vav1 protein lacking GEF activity clearly demonstrated that Vav1 enzymatic activity is not required for TCR-induced calcium flux and activation of the ERK MAPK pathway (15). Our previous genetic studies in Lewis (LEW) and Brown–Norway (BN) rat strains identified a major locus of 117-kb on rat chromosome 9 that controls the opposite susceptibility of those two strains to Th1 and Th2 immune-mediated diseases (7, 9, 16). Fine mapping of this locus identified a nonsynonymous exonic single nucleotide polymorphism in Vav1 leading to the substitution of an arginine by a tryptophan (R63W) in BN rats (7). Functional studies showed that the BN Vav1-W63 variant is constitutively active and exhibits normal GEF activity but reduced adaptor functions (7). Vav1-W63 variant was associated with a reduction of inflammatory cytokine production by T cells and decreased susceptibility to CNS inflammation (8).

Themis1 has been recently described as a new component of the TCR signaling machinery that controls thymic development of T cells. The ablation of Themis1 in C57BL/6 mice results in impaired T cell development, particularly of the CD4+ lineage (17–21). After TCR engagement, Themis1 is phosphorylated by Lck and Zap-70, and recruited to the transmembrane adaptor protein LAT (22, 23). Themis1 has been shown to interact with Vav1 and positively regulates its phosphorylation (23). However, it remains unclear whether Themis1 acts as a positive or negative regulator of TCR signaling (17, 23–25). We previously discovered a spontaneous mutation in the BN rat strain leading to the disruption of the Themis1 gene (10). This mutation leads to a defect in thymic development of CD4 T cells similar to that observed in Themis1-deficient C57BL/6 mice. Strikingly, the Themis1-deficient BN rats develop spontaneously inflammatory bowel disease (IBD) (10). The functional studies revealed that Themis1-deficient BN regulatory T cells (Tregs) migrate normally into the intestine but exhibit a defect in their suppressive functions. In contrast, no Treg dysfunction or spontaneous pathological manifestations have been reported in mouse models of Themis1 deficiency. We investigated in this study whether these differences reflect an epistasis phenomenon on the BN genetic background or species discrepancies.

We show in this article that the impact of Themis1 deficiency on Treg suppressive function and IBD development depends on the genetic background. In contrast with the BN rat strain, the disruption of the Themis1 gene in LEW rats does not lead to the spontaneous development of IBD or functional Treg defect, suggesting that an epistasis phenomenon in the BN genetic background controls these phenotypes. In addition, we show that the gene(s) responsible for this epistasis is (are) contained within the previously described 117-kb locus on rat chromosome 9 that controls susceptibility to immune-mediated diseases in LEW and BN rats (7, 9). Finally, a biochemical analysis of TCR signaling pointed out an epistasis between Themis1 and Vav1-W63 variant in the BN rats.

Materials and Methods

Animals

All breeding and experimental procedures were carried out in accordance with the European Union guidelines and were approved by our local ethical committee (license number: 31259). Rats were maintained under specific pathogen-free conditions in our facility. Specific pathogen-free BN and Lewis (LEW) rats were obtained from the Centre d’Elevage R. Janvier (Le Genest St. Isle, France). The BNThemis1−/− rats were bred from an autosomal recessive mutation that occurred spontaneously in a BN rat colony from our laboratory (10). The generation of the LEWThemis1−/− rat strain was performed according to the speed congenics strategy (26) as described previously (27). In brief, repeated backcrosses from (LEW×BNThemis1−/−) F1 hybrids to LEW rats were performed. In rats issued from the backcrosses, genomic composition was assessed using 200 polymorphic microsatellite markers selected to cover the whole genome with ∼10-cM intervals. At each backcross, selected recombinants carrying both the higher percentage of LEW genomic origin and the genomic interval from BN origin containing the mutated Themis1 gene were backcrossed to LEW rats. At the end of four successive backcrosses, all microsatellite markers showed homozygosis for LEW genome, but three markers on top of chromosome 1 extending on a 21-Mb interval, in the region bearing the Themis1 gene. Recombinants rats were then intercrossed to generate the LEWThemis1−/− rats that were homozygous for the Themis1 knockdown mutation from BN origin. The absence of Themis1 protein expression was confirmed by Western blot analysis (Supplemental Fig. 1A, 1B). The BN.LEWc9-Bf congenic line (Bf) was generated as described previously (7, 28). The BfThemis1−/− line was raised by intercrossing (BNThemis−/−×Bf) F1 rats and selecting recombinants carrying homozygosis for both the Themis1 mutation and the 117-kb interval from LEW origin that characterizes the Bf line. In this BfThemis1−/− line, Western blotting for Themis1 and Vav1 confirmed the absence of Themis1 and the increase in Vav1 protein amount that characterizes the LEW Vav1-R63 variant as compared with the BN Vav1-WR63 variant (Supplemental Fig. 1C, 1D). Themis1-deficient mice were kindly provided by Dr. Paul E. Love (National Institutes of Health, Bethesda, MD) and have been reported previously (17).

Results

Themis1 deficiency in the LEW genomic background reproduces the abnormalities of T cell thymic development observed in BNThemis1−/− rats but does not lead to the development of IBD

To investigate the impact of the genomic background on the pathophysiological consequences of Themis1 deficiency, we transferred the recessive autosomal Themis1 mutation from BNThemis1−/− rats to the LEW genomic background (Supplemental Fig. 1A) using the speed congenic strategy (27). The Themis1 disruption was confirmed by the absence of Themis1 protein expression in thymocytes (Supplemental Fig. 1B). We next investigated the thymic development of T cells in LEWThemis1−/− rats. Although there were no changes in CD4−CD8− double negative or CD4+CD8+ double positive thymocytes, the proportion (Fig. 1A) and absolute numbers (Fig. 1B) of CD4+ single-positive cells were greatly reduced in LEWThemis1−/− rats, together with a milder decrease in CD8+ single-positive thymocytes. Accordingly, the proportion (Fig. 1C) and absolute numbers (Fig. 1D) of CD4 T cells were markedly reduced in spleen (Fig. 1C, 1D) and lymph nodes of LEWThemis1−/− rats (data not shown). These abnormalities affected to a lesser extent the CD8 T cell compartment (Fig. 1C, 1D). Cohorts of LEWThemis1−/− rats were followed for 38 wk to analyze whether Themis1 deficiency in the LEW genetic background is accompanied by the development of pathological disorders. We did not observe any weight loss or clinical signs of spontaneous pathological manifestations. Furthermore, blinded histological analysis of numerous organs (salivary glands, stomach, kidneys, pancreas, skin, liver, pancreas, lungs, heart, brain, ovaries, testis, thyroid, intestines, and muscle) collected from 12- or 38-wk-old rats did not reveal any inflammatory cell infiltration in LEWThemis1−/− rats (data not shown). Thus, Themis1 disruption in the LEW genomic background reproduces the cardinal CD4 T cell lymphopenia phenotype described in BNThemis1−/− rats and in C57BL/6Themis1−/− mice (17–21). However, in contrast with what was observed in BN rats, this deficiency does not predispose to the development of spontaneous pathological diseases.

In LEW rats, Themis1 deficiency impacts on cytokine production by CD4 T cells but does not impair Treg suppressive functions

We first analyzed whether Themis1 deficiency affects splenic CD4 T cell function in a LEW genetic background. Consistent with our findings in BNThemis1−/− rats (10), we observed an overexpression of OX40 and a downregulation of CD62L expression in LEWThemis1−/− CD4 T cells (Fig. 2A), indicating an increased proportion of activated CD4 T cells. The cytokine production was then measured in both total and naive (CD62L+ CD25−) CD4 T cells after anti-TCR and anti-CD28 stimulation. As compared with CD4 T cells from LEW rats, both total (Fig. 2B) and naive CD4 T cells (Fig. 2C) from LEWThemis1−/− rats displayed increased production of IL-4, IL-10, and IL-17, thus confirming the skewed cytokine production previously associated with Themis1 deficiency in the BN genomic background. Of note, the IFN-γ production was not decreased by Themis1 deficiency in the LEW background, contrary to its effect in the BN background. Next, we investigated the impact of Themis1 deficiency on LEW Treg development and function. Although the proportion of Tregs among spleen CD4 T cells was increased in LEWThemis1−/− rats, the total number of Tregs was actually decreased, as a result of CD4 lymphopenia (Fig. 3A, 3B). We next studied Treg function in a coculture assay using CD4+ CD8− CD25bright T cells. Sorted Tregs from LEW and LEWThemis1−/− rats showed similar purity and expression levels of the Treg transcription factor Foxp3 (Fig. 3C, 3D). Interestingly, the Treg suppressive function was unaltered in LEWThemis1−/− rats (Fig. 3E, 3F). These observations concord with the absence of spontaneous disease because the development of IBD in BNThemis1−/− rats is linked to a functional defect of Tregs. Similarly, the suppressive function of Tregs purified from Themis1-deficient C57BL/6 mice was similar to that of Tregs originating from wild-type mice (Supplemental Fig. 2). Thus, the differential consequence of Themis1 deficiency observed between BNThemis1−/− rats and mice rather reflects an epistatic phenomenon occurring in the BN genetic background than species discrepancies.

Themis1 deficiency in LEW rats does not alter Treg suppressive functions. (A) Proportions of CD25high and Foxp3+ cells among CD4 T cells from spleens of LEW (n = 4) and LEWThemis1−/− (n = 4) rats. (B) Absolute numbers of Foxp3+ CD4+ T cells in spleen of LEW and LEWThemis1−/− rats. (C) Percentages of Foxp3+ cells and (D) Foxp3 expression levels among sorted CD25bright CD4+ CD8− TCR+ Tregs used in coculture experiments. Data are presented as mean ± SEM. (E and F) Suppressive activity of CD25bright CD4+ T cells from LEW and LEWThemis1−/− rats assessed in coculture experiments with CellTrace-labeled naive LEW CD4+ T cells as effector cells stimulated with plate-bound anti-CD3 and irradiated syngeneic APC. For each experiment, we pooled the purified Tregs from three rats per group because of the low numbers of Tregs present in Themis1-deficient rats. (E) Representative histograms of the proliferation of effector cells alone or in the presence of LEW and LEWThemis1−/− Tregs (Treg/Teff ratio 1:2). Percentages indicate the proportion of CellTrace low effector cells. Results are representative of one of three independent experiments. (F) Suppressive activity of CD25bright CD4+ T cells from LEW and LEWThemis1−/− rats at different Treg/Teff ratios. Data are expressed as percentage of inhibition and presented as mean values ± SEM obtained from three independent experiments. *p ≤ 0.05. ns, not significant.

The introgression of the 117-kb interval bearing the LEW Vav1-R63 variant in BN rats restores the suppressive function of Themis1-deficient Tregs

The striking differences in the impact of Themis1 deficiency in BN or LEW genomic backgrounds raised the hypothesis that an epistasis phenomenon occurs in the BN rats. Thus, a peculiar BN gene(s) variant(s) could be required to confer the Treg defect and IBD development in the absence of Themis1. The TCR signaling molecule Vav1 could be the gene responsible for this epistatic phenomenon for the following reasons: 1) BN rats harbor a peculiar Vav1 variant (Vav1-W63) with reduced adaptor function and signaling capacities (7), and 2) Themis1 has been shown to interact and to positively regulate Vav1 phosphorylation in thymocytes (23). Therefore, because Themis1 is also expressed on rat Tregs (data not shown), the effect of Themis1 deficiency on BN Treg function could result from a cumulative impairment of the TCR signaling due to the association of Themis1 deficiency with the presence of the Vav1-W63 variant. To test this hypothesis, we crossed BNThemis1−/− rats (bearing the Vav1-W63 variant) with BN congenic rats bearing a 117-kb interval from LEW origin containing the wild type Vav1-R63 variant (congenic line named Bf) to generate the BfThemis1−/− rats. Those BfThemis1−/− rats of BN genomic background carry both the Themis1 deficiency and the Vav1-R63 variant (Supplemental Fig. 1C, 1D).

As observed in BNThemis1−/− and LEWThemis1−/−, BfThemis1−/− rats also showed an impaired thymic T cell development leading to a peripheral T cell lymphopenia, an increased frequency of activated peripheral CD4 T cells, and a biased cytokine production by total or naive CD4 T cells (Supplemental Fig. 3). We then investigated whether the differential impact of Themis1 deficiency on Treg functions observed between the BN and LEW strains depend on the 117-kb interval containing the LEW Vav1-R63 variant by analyzing Treg functions in the BfThemis1−/− strain. First, we controlled that Themis1 deficiency had no impact on the percentage of Foxp3-expressing cells among TCR+ CD4+ CD8− CD25bright sorted Tregs, nor on the level of Foxp3 expression (Fig. 4A, 4B), irrespective of the Vav1 variant. Then, using coculture experiments, we compared the suppressive activity of Tregs from BN and BNThemis1−/− (Vav1-W63 variant), on the one hand, and of Bf and Bf Themis1−/− (Vav1-R63 variant), on the other hand. As previously described, a clear defect in the suppressive activity of BNThemis1−/− Tregs was found. By contrast, under the same experimental conditions, BfThemis1−/− Tregs exhibited an intact in vitro suppressive activity as compared with Bf Tregs (Fig. 4C). These results reveal that the defect in Treg suppressive activity associated with Themis1 deficiency depends on the 117-kb interval containing the Vav1-W63 polymorphism. This defect concerns natural Tregs but not induced Tregs because BN and BN Themis1−/− Tregs generated in the presence of TGF-β exhibit similar suppressive activity (Supplemental Fig. 4).

Effect of Themis1 deficiency on Treg functions depends on the 117-kb interval containing Vav1 variants. (A) Percentage of Foxp3+ cells and (B) Foxp3 expression levels in sorted CD25bright CD4+ CD8− TCR+ Tregs used in coculture experiments. (C) Suppressive activity of CD25bright CD4+ T cells from BN and BNThemis1−/− rats (left) or Bf and BfThemis1−/− rats (right) assessed in coculture experiments with CellTrace-labeled naive effector cells. Representative histograms of one experiment of four independent experiments are shown. The suppressive activity of CD25bright CD4+ T cells at different Treg/Teff ratios is expressed as percentage of inhibition of the response of effector cells alone. Results are presented as mean values ± SEM obtained from four independent experiments, each experiment including pools of four rats per group. *p ≤ 0.05. ns, not significant.

In BNThemis1−/− rats, the introgression of the 117-Kb interval bearing the LEW Vav1-R63 variant protects from IBD development

Because the Treg defect observed in the BNThemis1−/− is linked to the development of an IBD, we reasoned that BfThemis1−/− rats that harbor Tregs with normal suppressive function would not develop signs of inflammation. To test this hypothesis, we examined organs from BfThemis1−/−, BNThemis1−/−, and age-matched control Bf and BN rats. BfThemis1−/− rats were devoid of tissue inflammation, whereas BNThemis1−/− rats showed inflammation of the intestinal tract as revealed by the macroscopic lesions including edema, erythema, and increased mucous secretion. The disease prevalence rate reached 51% in BNThemis1−/− rats, whereas only 1 BfThemis1−/− rat out of 46 showed minor signs of inflammation (Fig. 5A). Histopathological studies (Fig. 5B, 5C) and quantitation of MPO activity in the various parts of the intestinal tract (Fig. 5D) confirmed that the introduction of the 117-kb interval from LEW origin protected BNThemis1−/− rats from IBD development. Thus, as anticipated, the restoration of the suppressive function of Tregs in Themis1-deficient rats bearing the 117 kb of LEW origin is accompanied by the absence of IBD.

A previous study showed that Themis1 could interact with Vav1 (23). Because Vav1 appeared to be the strongest candidate gene responsible for the epistasis, we investigated whether Vav1-R63 and Vav1-W63 variants interact with Themis1 differently using cotransfection experiments in HEK cells. The obtained results reveal that the BN Vav1-W63 variant interacts more efficiently with Themis1 as compared with the Vav1-R63 variant (Fig. 6A, 6B). Next, we investigated the impact of Themis1 deficiency on the recruitment of Vav1 to the LAT platform after TCR stimulation using thymocytes. This analysis revealed that the absence of Themis1 reduced the recruitment of the Vav1-W63 variant from BN origin to LAT (Fig. 6C, 6D), whereas it had no impact on the recruitment of Vav1-R63 from LEW origin. However, despite this reduced recruitment, the phosphorylation of the Vav1-W63 variant was not affected by Themis1 deficiency (Fig. 6E). In cell expressing Vav1-W63 variant, Themis1 deficiency led to a decreased recruitment of PLC-γ and Grb2 to LAT, whereas it did not exert such an effect in Vav1-R63 expressing cells (Fig. 6C, 6D). We next analyzed the consequences of this modified proximal TCR signaling complex assembly on the downstream signaling pathways Erk and Akt, known to be respectively dependent on Vav1 adaptor function and enzymatic activity (15). Themis1 deficiency reduced Erk activation in cells expressing the Vav1-W63 variant but not in those expressing Vav1-R63 (Fig. 6E). In contrast, Akt activation was not affected by Themis1 deficiency. Together, these results show that Themis1 interacts more efficiently with Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT to fully promote the activation of Erk kinases that are likely necessary for the suppressive functions of Tregs (29–31).

Association of Themis1 deficiency and Vav1-W63 polymorphism impacts on LAT signalosome and impairs TCR-induced Erk signaling. (A) Western blot analysis of Vav1-W63 or Vav1-R63 interaction with Themis1. c-Myc–tagged Vav1 variants and flag-tagged Themis1 were overexpressed in HEK cells, and Themis1 were immunoprecipitated in untreated or pervanadate-stimulated HEK cells. (B) Densitometric quantification of Vav1 after Themis1 immunoprecipitation; relative intensities are normalized to the respective Themis1 signal. (C) Western blot analysis of Vav1, PLC-γ, Grb2, and LAT after LAT immunoprecipitation in unstimulated thymocytes (left) or after 1 min anti-TCR/anti-CD4 stimulation (right). Protein levels in whole cell lysates (WCL) are shown in the lower panel. (D) Densitometric quantification of Vav1, PLC-γ, and Grb2 after LAT immunoprecipitation; relative intensities normalized to the respective LAT signal are shown. (E) Western blot analysis of P-Erk, P-Vav1, P-Akt activity, and total Vav1, Erk, and Akt protein levels in anti-TCR/anti-CD4–stimulated thymocytes. Results shown are representative of two independent experiments.

Discussion

Tregs play a pivotal role in maintaining immunological self-tolerance and immune system homeostasis. They are defined by the expression of Foxp3, a specific transcription factor necessary for their development and function (32–36). However, there is substantial evidence that Foxp3 is not the sole cell-intrinsic regulator involved in the maintenance of Treg suppressive function (37, 38). In this context, we previously reported that deficiency for the TCR signaling molecule Themis1 in BN rats impairs the suppressive function of Tregs, despite normal levels of Foxp3 expression, and leads to the spontaneous development of IBD (10). In addition, we showed that these Tregs migrate normally in the intestine, thus excluding the involvement of Treg migration defect in this phenotype (Supplemental Fig. 5).

In this study, we show that the pathophysiological consequence of Themis1 deficiency depends on another gene that plays key roles in TCR-dependent signaling. Indeed, Themis1-deficient LEW rats, although lymphopenic, did not develop IBD or other spontaneous diseases and their Tregs were fully functional. These results exclude lymphopenia as the factor responsible for the Treg defect in Themis1-deficient BN rats and favor the hypothesis of an intrinsic role of Themis1 in controlling Treg function. The striking differences observed in Themis1-deficient rats of the BN and LEW genetic backgrounds suggest that an epistasis between Themis1 and other gene(s) controls Treg function. We showed that the 117-kb locus on rat chromosome 9 previously implicated in modulating the susceptibility of LEW and BN rats to immune-mediated diseases (7, 9) contains the gene involved in this epistasis. Indeed, the functional defect of Tregs and the spontaneous IBD development were observed only in rats that combine Themis1 deficiency with the presence of the 117-kb locus of BN origin. This 117-kb region contains only four genes: C3 (complement 3 precursor), Gpr108 (G protein–coupled receptor 108), Cip4/Trip10 (Cdc42-interacting protein 4), and exons 1–15 of Vav1. The coding regions of these four genes were previously sequenced and led to the identification of only two nonsynonymous polymorphisms in exon 32 of C3 and in exon 1 of Vav1 (7). Vav1 appeared to be a strong candidate gene responsible for the epistasis in the BN rats for the following reasons. First, Themis1 and Vav1 are components of the same SLP76-LAT signalosome (22, 23, 39, 40). Second, Vav1 phosphorylation is decreased in Themis1-deficient mice after TCR engagement (23), suggesting that Themis1 is a positive regulator of Vav1. Third, the BN Vav1 contains a polymorphism (R63W) responsible for the reduction of its adaptor function (7). Fourth, the BN Vav1-W63 variant interacts more efficiently with Themis1 as compared with the LEW Vav1-R63 variant. Finally, we show that Themis1 deficiency reduces the recruitment of Vav1-W63 to LAT signalosome and impairs TCR signaling as revealed by the defect in Erk activation. In contrast, Themis1 deficiency has no apparent effect on ERK activation in rat thymocytes containing the Vav1-R63 variant, consistent with findings obtained in Themis1-deficient C57BL/6 mice, which harbor the Vav1-R63 variant (19). Thus, this study suggests that Themis1 and Vav1 form a cooperative signaling complex important for the optimal transmission of TCR-mediated signals necessary for Tregs to mediate their suppressive functions.

Besides the functional Treg defect observed in Themis1-deficient BN rats, we previously reported that effector CD4 T cells secreted less IFN-γ and higher amounts of Th2/Th17 cytokines (10). This effect may be directly related to the role played by Themis1 in TCR signaling in effector T cells or to a secondary effect of lymphopenia induced by Themis1 deficiency. In this article, we show that Themis1-deficient LEW and Bf CD4 T cells produce exacerbated Th2/Th17 cytokines, whereas their production of IFN-γ was not impacted by Themis1 deficiency. These data suggest that the cooperation between Themis1 and Vav1-W63 is mandatory for IFN-γ production, whereas the exacerbated production of Th2/Th17 cytokines might be secondary to Themis1 deficiency–induced lymphopenia. In this regard, it has been shown that lymphopenia may influence the orientation of immune responses by affecting the differentiation of T cell subsets (41, 42). Thus, depending on the genetic background, Themis1 might influence the development of immune-mediated diseases by affecting both Treg suppressive functions and the production of IFN-γ by effector CD4 T cells.

The mechanism whereby the combined presence of Vav1-W63 variant and Themis1 deficiency lead to functional defect of Tregs and reduced IFN-γ production by effector CD4 T cells is still unknown. We previously showed that the Vav1-W63 variant is constitutively active and exhibits normal GEF activity but reduced adaptor functions associated with a marked reduction in Vav1 protein levels (7). In this study, we showed that Themis1 interacts more efficiently with Vav1-W63, and this may promote proximal TCR signaling assembly to compensate for the signaling defect related to Vav1-W63. Indeed, we showed that Themis1 favors Vav1-W63 recruitment to LAT and this was associated with an increased Grb2 and PLC-γ recruitment to this signaling platform. In Themis1-deficient BN rats, we observed a reduction of Vav1-W63 recruitment to LAT that is likely responsible for the defect in Erk activation, because Erk activity depends on Vav1 adaptor function (15). Of note, in the absence of Themis1 in BN rats, PLCγ and Grb2 recruitment to LAT remains in a similar range compared with the Bf strain. These results indicate that the compensatory effect of Themis1 in the presence of the W63-Vav1 might involve, besides PLCγ and Grb2, other molecules to stabilize the LAT signalosome complex. This study revealed an association among the reduced Vav1-W63 recruitment to the LAT signalosome, the defective Erk activity, the Treg functional defect, and the decreased production of IFN-γ by effector T cells. These data are consistent with previous reports showing the critical role of the integrity of LAT signaling platform as well as Erk activation for the Treg suppressive function and the Th1/Th2 polarization (29–31, 43, 44). Indeed, the disruption of LAT/ PLC-γ signalosome assembly reduces Erk and Ca2+ activation (29, 30) and despite normal Foxp3 expression, LAT- or PLC-γ–deficient Tregs are ineffective in controlling the proliferation of effector T cells (29–31). In addition, sustained Erk activity and calcium signaling impact on the Th1/Th2 polarization, promoting Th1 differentiation (43–45). Finally, it has been shown that TCR-dependent signals promote epigenetic modifications (46) that are crucial for the acquisition of a functional and stable Treg phenotype by developing Foxp3+ cells (47). It remains to be determined whether the functional defect of Tregs in Themis1-deficient BN rats depends on epigenetic modifications induced by the reduction of TCR signaling during Treg development.

This study describes for the first time, to our knowledge, the importance of the cooperation between Themis1 and Vav1 for the control of Treg suppressive function and the development of a spontaneous inflammatory disease. The epistatic interaction between Themis1 and Vav1 could have important repercussions in genetics of human pathologies. Indeed, Vav1 has been pointed out as a susceptibility gene for multiple sclerosis (8). Themis1 also has been identified as a potential candidate for Crohn’s disease (48), celiac disease (49–51), and multiple sclerosis (52). Given the strong impact of the Themis1/Vav1 cooperation revealed by our study, it remains to be tested whether the association of variants of those two genes could exert synergetic effects on susceptibility to immune-mediated pathologies.

In conclusion, our study highlights the importance of studying gene–gene interactions in complex diseases and provides insights into the underlying cellular and molecular mechanisms. Given the large number of genome-wide association studies that have recently been performed, it is clear that genome-wide interaction testing will be the logical next step after single-locus testing. This will require the development of new powerful bioinformatics tools that take into account gene–gene interactions in GWAS data. Such approaches should become possible in the coming years (53–55) and are likely to generate novel advances in our understanding of the genetic component in complex diseases.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank P. Rousset for technical help with MPO assays; D. Dunia, R. Liblau, and L. Mars for comments on the manuscript; and the flow cytometry core facility (Centre de Physiopathologie de Toulouse Purpan), the animal house staff, and the histopathology core facility for assistance (Inserm US006 ANEXPLO/CREFRE). This work is dedicated to the memory of our mentor Prof. Philippe Druet, who recently died.